Method for determining polynucleotides in a sample without attaching these to a support, and using detection probes

ABSTRACT

The invention relates to a method for detecting polynucleotides on a substrate, whereby the substrate contains a multitude of separate detection regions each accommodating different polynucleotides. The polynucleotides in the detection regions exist, contrary to known DNA chip test formats, in a form that is not bound to the substrate. The invention also relates to a device for implementing said method.

The invention relates to a method for detecting polynucleotides on asupport which contains a multiplicity of separate detection regions within each case different polynucleotides. In contrast to known DNA-chiptest formats, the polynucleotides in said detection regions are notbound to said support. Furthermore, an apparatus for carrying out themethod is disclosed.

The use of “DNA chips” for determining polynucleotides is known (see,for example, European patents EP 0 373 203 and 0 619 321). DNA chips ofthe prior art contain a multiplicity of separate detection regions onthe surface of a support, which contain in each case differentpolynucleotides in a support-bound form. However, these DNA chips have adisadvantage in that the reactivity of the polynucleotides immobilizedon the support differs substantially from the reactivity of freepolynucleotides, as are present in biological systems. This leads tofundamental problems when evaluating hybridization experiments usingcommon DNA chips.

It was the object of the present invention to provide novel methods fordetermining polynucleotides, whose efficiency, on the one hand, iscomparable to that of common DNA chips but which, on the other hand,eliminate the disadvantages which the use of immobilized polynucleotidesentails.

This object is achieved by a method for determining polynucleotides,comprising the steps:

-   -   (i) providing /a support containing a plurality of separate        detection regions each of which contains a polynucleotide which        is not bound to said support,    -   (ii) contacting the polynucleotides located in said detection        regions with detection probes, with individual detection regions        each containing different combinations of polynucleotide and        detection probe, and    -   (iii) determining a hybridization of said polynucleotides with        said detection probes.

A substantial advantage of the method of the invention is thepossibility of hybridizing the polynucleotides to be studied withdetection probes in very small measuring volumes and at very lowconcentrations of the reaction partners. To this end, preference isgiven to using the method of fluorescence correlation spectroscopy (FCS)described in European patent 0 679 251 or another fluorescencespectroscopy method with sufficient sensitivity. Said FCS preferablycomprises measuring one or a few sample molecules in a measuring volume,the concentration of the molecules to be determined being ≦10⁻⁶ mol/land the measuring volume being preferably ≦10⁻¹⁴ l. Fluorescencecorrelation spectroscopy comprises determining substance-specificparameters which are obtained by luminescence measurements on themolecules to be determined. These parameters may be translationaldiffusion coefficients, rotational diffusion coefficients or/and theexcitation wavelengths, the emission wavelengths or/and the lifetime ofan excited state of a luminescent substituent or the combination of saidparameters. For details of carrying out the method and details of theapparatuses used for said method, reference is made to the disclosure ofEuropean patent 0 679 251.

As an alternative, it is also possible to carry out a fluorescence decaymeasurement in which the relaxation time of the fluorescent label or theoccurrence of energy transfer or quenching processes is determined.

The polynucleotides arranged in the detection regions of the support arepreferably nucleic acids such as DNA or RNA, DNA polynucleotides beingparticularly preferred. On the other hand, it is also possible to usenucleic acid analogs such as, for example, peptide nucleic acids (PNA)as polynucleotides. On the one hand, the polynucleotides may be derivedfrom natural resources, for example from gene or cDNA libraries and maybe genes, cDNA molecules or fragments thereof but, on the other hand,they may also be synthetically generated, for example combinatorial,polynucleotide sequences. The polynucleotides are in a form which canhybridize with a complementary detection probe under the conditions ofthe detection method. The polynucleotides are preferablysingle-stranded.

Preferably the support contains a plurality of detection regions each ofwhich contains a polynucleotide with differing sequence. However, it isalso possible to use a support having a plurality of detection regionswhich in each case contain a polynucleotide with identical sequence. Inthis case, a different detection probe must be employed for each ofthese detection regions. The length of the polynucleotides in thedetection regions is preferably at least 10 nucleotides to severalthousand nucleotides.

The detection probes used for studying a hybridization with thepolynucleotides located in the detection regions may, like thepolynucleotides themselves, be derived from natural or syntheticsources. The detection probes preferably carry one or more labelinggroups which may be introduced during synthesis of the detection probes(in particular in chemical synthesis or in amplification) or else aftersynthesis of the detection probes (for example via enzymatic attachmentto the 5′ or 3′ end). Thus it is possible to generate labeled detectionprobes, for example, via reverse transcription of RNA molecules, forexample of mRNA molecules, by using labeled primers or via enzymaticprimer extension by using labeled nucleotide building blocks.

In a particularly preferred embodiment of the invention, polynucleotidescoupled to a microparticle are used. The size of said microparticle issufficient for slowing down the rate of diffusion of a polynucleotidecoupled thereto. On the other hand, however, the microparticle is alsosufficiently small in order to ensure that the reaction behavior of thepolynucleotide coupled thereto essentially corresponds to that of a“free” but not an “immobilized” polynucleotide. The microparticle sizeis preferably ≦1 μm, particularly preferably 10 nm to 500 nm. Themicroparticles may be latex particles, for example of polystyrene, orother polymers, metal sol particles, silica particles, quartz particlesor glass particles. Polynucleotides can bind to microparticles viacovalent or noncovalent interactions. For example, binding of thepolynucleotide to the microparticle can be mediated by high-affinityinteractions between the partners of a specific binding pair, forexample biotin/streptavidin or avidin, hapten/anti-hapten antibody,sugar/lectin, etc. Thus it is possible to couple biotinylatedpolynucleotides to streptavidin-coated microparticles. As analternative, it is also possible to bind the polynucleotides toparticles via adsorption. Thus it is possible to bind polynucleotidesmodified by incorporation of alkanethiol groups to metal particles, forexample gold particles. Yet another alternative is covalentimmobilization in which binding of the polynucleotides can be mediatedvia reactive silane groups on a silica surface. It is also possible,where appropriate, to use alternatively or additionally detection probescoupled to microparticles.

A further possibility is to prepare the polynucleotides or detectionprobes by support-based synthesis on the particle by using knownsolid-phase synthesis methods.

A single polynucleotide strand may be bound to a microparticle. However,it is also possible to bind a plurality of, for example, up to severalthousand, polynucleotide strands to a particle. Binding takes placepreferably via the 5′ or the 3′ terminals of the polynucleotide strand.

The method of the invention uses a support with a plurality of,preferably with a multiplicity of 10² or more, separate detectionregions. The detection regions are designed in such a way that theyenable hybridization of the polynucleotide with the detection probe insolution. The detection regions are preferably depressions in thesupport surface, it being in principle possible for said depressions tohave any shape, for example circular, square, diamond-shaped, etc. Thevolume of the detection regions is preferably ≦10⁻⁶ l and particularlypreferably ≦10⁻⁸ l. The support preferably contains 10³ or more,particularly preferably 10⁴ or more, separate detection regions.

Preferred concentrations of the polynucleotides to be determined in thedetection regions are ≦10⁻⁶ mol/l, particularly preferably 10⁻¹⁰ to10⁻¹⁴ mol/l, while the detection probes are supplied at a concentrationof preferably ≦10⁻⁴ mol/l, particularly preferably 10⁻⁸ mol/l to 10⁻¹¹mol/l.

The method of the invention preferably uses labeled detection probes,labels detectable via luminescence measurements, in particular labelsdetectable via fluorescence measurement, being preferred. When thedetection probe used in a detection region hybridizes with thepolynucleotide located there, this hybridization causes a detectablechange in a substance-specific parameter. This detectable change can bemeasured via fluorescence measurement. Thus, for example, in the case ofhybridization of detection probe and polynucleotide, the mobility of thehybrid is detectably reduced compared with the starting components. Asan alternative or in addition, it is also possible to detect theoccurrence of quenching processes or energy transfer processes duringhybridization. Such energy transfer processes occur, for example, whenusing a plurality of different labeled probes or labeled polynucleotidesand labeled probes.

Thus, for example, detection may be carried out by means of confocalsingle-molecule detection, for example via fluorescence correlationspectroscopy according to EP 0 679 251, for which a very small,preferably confocal, volume element, for example from 0.1×10⁻¹⁵ to20×10⁻¹² l, is exposed to the excitation light of a laser, which excitesthe fluorescently labeled detection probes located in this measuringvolume so that they emit fluorescence light, the emitted fluorescencelight being measured by means of a photodetector and a correlation beingdetermined between the change in the measured emission with time and themobility of the fluorescent labeling group. The confocal determinationof single molecules is furthermore described in Rigler and Mets (Soc.Photo-Opt. Instrum. Eng. 1921 (1993), 239 ff.) and Mets and Rigler (J.Fluoresc. 4 (1994), 259-264).

As an alternative or in addition, detection may also be carried out viatime-resolved decay measurement, so-called time gating, as described,for example, by Rigler et al.: Picosecond Single Photon FluorescenceSpectroscopy of Nucleic Acids, in: “Ultrafast Phenomena”, D. H. Austoned. Springer 1984. In this case, the fluorescent molecules are excitedin a measuring volume followed by, preferably at a time interval of ≧100ps, opening a detection interval on the photodetector. In this way it ispossible to keep background signals generated by Raman effectssufficiently low so as to make possible an essentially interference-freedetection. Time gating is particularly suitable for measuring quenchingprocesses or energy transfer processes.

In a preferred embodiment of the method, the determination may alsocomprise measuring a cross-correlated signal derived from aprobe-polynucleotide complex containing at least two different labels,in particular fluorescent labels, it being possible to use a pluralityof differently labeled probes or/and polynucleotides with in each casedifferent labels. This cross-correlation determination is described, forexample, in Schwille et al. (Biophys. J. 72 (1997), 1878-1886) andRigler et al. (J. Biotechnol. 63 (1998), 97-109).

The support used for the method should be designed so as to makepossible optical detection in the detection regions. Thereforepreference is given to using a support which is optically transparent atleast in said detection regions. The support may either have totaloptical transparency or contain an optically transparent base and anoptically impermeable cover layer with gaps in the detection regions.Examples of materials suitable for supports are composite supports madefrom metal (e.g. silicon for the cover layer) and glass (for the base).Supports of this type can be generated, for example, by applying a metallayer with predetermined gaps for the detection regions to the glass. Asan alternative, it is possible to use plastic supports, for example ofpolystyrene, or polymers based on acrylate or methacrylate.

The invention further relates to an apparatus for determiningpolynucleotides, comprising a support containing a plurality of separatedetection regions each of which contains a polynucleotide which is notbound to the support. The apparatus may be prepared by introducing thepolynucleotides into the detection regions of the support in the form ofa solution, preferably an aqueous solution, and subsequent drying. Theapparatus generated in this way is stable for a long time, for exampleseveral months.

The invention still further relates to a kit of reagents for determiningpolynucleotides, comprising a device of the invention and (a) a set oflabeled detection probes or (b) means for preparing a set of labeleddetection probes, for example labeled primers or labeled(deoxy)ribonucleoside triphosphates, and, where appropriate, enzymes forpreparing suitable detection probes.

The method of the invention may be used, for example, for functionalgenomics and for transcriptome analysis.

BRIEF DESCRIPTION OF THE FIGURES

Furthermore, the present invention is intended to be illustrated by thefollowing figures and examples in which:

FIG. 1: shows the diagrammatic representation of a support (2) suitablefor carrying out the method of the invention, having a multiplicity ofdetection regions (4) formed as depressions on said support. A supporthaving an area of from 1 to 2 cm² may contain, for example, up to 10⁴depressions.

FIG. 2: shows a cross section through a support of the invention. Thesupport contains a base (6), preferably made from an opticallytransparent material such as glass, and a cover layer (8) with gaps, forexample made from silicon. Two detection regions (4 a, 4 b) are shownwhich contain in each case a polynucleotide (10 a, 10 b) coupled to amicroparticle and a detection probe (12 a, 12 b). In the detectionregion (4 a) the polynucleotide (10 a) hybridizes with the detectionprobe (12 a), while in the detection region (4 b) no hybridizationbetween the polynucleotide (10 b) and the detection probe (12 b) takesplace. A hybridization between microparticle-coupled polynucleotide (10a) and labeled detection probe (12 a) can be detected by studying singlemolecules.

It is possible to use for detection a detection apparatus (14) which ispreferably arranged below the support base. The detection apparatus (14)may contain a laser and a detector. Preference is given to using alaser-detector matrix consisting of a dot matrix of laser dots generatedby diffraction optics or a quantum well laser and of a detector matrixgenerated by fiber-coupled individual avalanche photodiode detectors oran avalanche photodiode matrix. Alternatively, it is also possible touse an electronic detector matrix, for example a CCD camera.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE

In a matrix of m×n microdepressions on one or more supports, a libraryof 4^(n) different nucleotide sequences of length m is generated, andeach sequence is transferred to a separate detection region. Thenucleotide sequences contain a biotin group on their 5′ end and arecoupled to streptavidin-coated microparticles.

An mRNA or cDNA library is provided with fluorescently labelednucleotides at the 3′ and/or 5′ end by an enzymatic reaction, forexample using terminal transferase or ligase. The labeled nucleotidesare pipetted into the depressions of the support.

Specific hybridization between polynucleotide and a labeled detectionprobe is carried out by analyzing the translation movement usingconfocal single-molecule analysis. Detection is carried out via a laserdetector matrix.

1. A method for determining a target polynucleotide, comprising:providing a support containing a plurality of separate detectionregions, each of which contains a target polynucleotide to be determinedwhich is neither directly nor indirectly bound to said support;contacting the target polynucleotide located in each of said detectionregions with a labeled detection probe, with individual detectionregions each containing different combinations of hybridizing targetpolynucleotide and labeled detection probe to form hybrid moleculesbetween said target polynucleotide and said labeled detection probe; anddetermining hybridization of said target polynucleotide with saidlabeled detection probe by detecting reduced mobility of hybridmolecules containing target polynucleotide and labeled detection probe,compared with non-hybrid molecules, wherein said target polynucleotideis coupled to a non-immobilized microparticle having a size of ≦1 μm,wherein said non-immobilized microparticle remains in solution duringdetermination of hybridization.
 2. The method of claim 1, wherein thepolynucleotide concentration in the detection regions is ≦10⁻⁶ mol/l. 3.The method of claim 1 wherein the volume of the detection regions is ineach case ≦10⁻⁶1 or less.
 4. The method of claim 1 wherein the supportcontains at least 10³ or more separate detection regions.
 5. The methodof claim 1, wherein said labeled detection probe is a fluorescent label.6. The method of claim 1, wherein said determining step is carried outby confocal single-molecule detection.
 7. The method of claim 6, whereinsaid determining step is carried out by at least one method selectedfrom the group consisting of fluorescence correlation spectroscopy andtime-resolved decay measurement.
 8. The method of claim 1, wherein thedetermining step comprises detecting the mobility of at least one ofsaid target polynucleotide or said labeled detection probe.
 9. Themethod of claim 1, wherein the determining step comprises detecting theoccurrence of quenching processes or energy transfer processes.
 10. Themethod of claim 1, wherein the determining step comprises measuring across-correlated signal which is derived from a labeled probe-targetpolynucleotide complex containing at least 2 different labels.
 11. Themethod of claim 10, wherein said at least two different labels arefluorescent labels.
 12. The method of claim 1, wherein said support isoptically transparent at least in a detection region.
 13. The method ofclaim 1, wherein said support comprises an optically transparent baseand an optically impermeable cover layer with gaps in the detectionregions.
 14. The method of claim 1, wherein said support comprises atleast one material selected from the group consisting of metal, glassand plastic.
 15. The method of claim 1, wherein said non-immobilizedmicroparticle has a size of from 10 nm to 500 nm.